Anaemia is a major global health problem arising from diverse causes and for which improved therapeutic strategies are needed. Erythroid cells can undergo apoptotic cell death and loss of ...pro-survival BCL-XL is known to trigger apoptosis during late-stage erythroid development. However, the mechanism by which loss or pharmacological blockade of BCL-XL leads to erythroid cell apoptosis remains unclear. Here we sought to identify the precise stage of erythropoiesis that depends on BCL-XL. We also tested whether deficiency of BIM or PUMA, the two main pro-apoptotic antagonists of BCL-XL, could prevent reticulocyte death and anaemia caused by BCL-XL loss. Using an in vivo mouse model of tamoxifen-inducible Bclx gene deletion and in vitro assays with a BCL-XL-selective inhibitor, we interrogated each stage of erythrocyte differentiation for BCL-XL dependency. This revealed that reticulocytes, but not orthochromatic erythroblasts, require BCL-XL for their survival. Surprisingly, concurrent loss of BIM or PUMA had no significant impact on the development of anemia following acute BCL-XL deletion in vivo. However, analysis of mixed bone marrow chimaeric mice revealed that loss of PUMA, but not loss of BIM, partially alleviated impaired erythropoiesis caused by BCL-XL deficiency. Insight into how the network of pro-survival and pro-apoptotic proteins works will assist the development of strategies to mitigate the effects of abnormal cell death during erythropoiesis and prevent anaemia in patients treated with BCL-XL-specific BH3-mimetic drugs.
Abstract
RAS proteins are membrane bound GTPases that when hyperactivated, act as oncogenes through activation of the MAPK & PI3K pathways. Each of these pathways has oncogenic potential, as ...exemplified by the high frequency of BRAF mutations in melanoma and PIK3CA and PTEN mutations in breast and gynecological cancers. Simultaneous activation of these pathways, as occurs in RAS driven cancers, generates aggressive cancers that present a significant clinical challenge. KRAS, the most commonly mutated RAS isoform, is also the most frequently mutated oncogene in cancer. While treatment options have improved for a subset of these patients due to the accelerated approval of KRAS-G12C inhibitors, the rapid development of resistance highlights the continued need for effective treatments. In RAS driven cell and animal models, dual inhibition of the MAPK &PI3K pathways has shown superior efficacy relative to targeting the individual pathways, however dose limiting toxicities in humans have prevented this combination strategy from finding clinical success. While physiological activation of the MAPK pathway is RAS dependent, the interaction between RAS and the catalytic subunit of PI3Kα, PIK3CA, serves as an amplifier but not a primary activator of this pathway. This interaction is particularly important in cancerous cells as it serves to amplify basal PI3K activity and support tumor progression. Conversely, in healthy cells, RAS independent activation of PI3K by upstream signaling factors is sufficient for maintaining physiological homeostasis. Unfortunately, traditional strategies of targeting the PI3K pathway have been unable to capitalize on this therapeutic opportunity as they do not discriminate between RAS dependent and RAS independent signaling. This leads to on-target dose-limiting toxicities, most commonly hyperglycemia and rash. We have discovered small molecules that disrupt the RAS:PIK3CA interaction through covalent ligation of C242, in the RAS binding domain, adjacent to the RAS binding interface. Using a Nanobit system to measure the RAS:PIK3CA interaction and signaling assays in “RAS active” cells, we have optimized molecules that disrupt the RAS:PIK3CA interaction and inhibit RAS mediated activation of PIK3CA. These molecules show robust inhibition of PIK3CA signaling, and tumor growth inhibition in cancer models, but unlike PIK3CA active site inhibitors, they do not impact glucose handling. Profound efficacy is seen when these RAS:PIK3CA inhibitors are used in combination with an agent targeting the MAPK pathway or with a therapy directly targeting mutant KRAS. Finally, we found that ligation of C242 on PIK3CA blocks HER2/3 driven activation of PI3Kα in a RAS independent manner. Overall, our data supports the clinical investigation of these molecules, particularly in combination with rationally chosen therapies where they may provide a tolerable and efficacious means of blocking the PI3K pathway.
Citation Format: Joseph Klebba, Junko Tamiya, Jinwei Wang, Hui Miao, Kelsey Lamb, Aaron Snead, Antonio Esparza-Oros, Richard Lin, Chanyu Yue, Stephanie Grabow, Steffen Bernard, Yongsheng Liu, Benjamin Horning, Melissa Hoffman, Marie Pariollaud, Taylor Wyrick, Holly Parker, John Sigler, Martha Pastuszka, David Weinstein, Todd Kinsella, Matt Patricelli. Development and characterization of covalent inhibitors of the RAS-PIK3CA interaction abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6501.
Antibody-mediated tumour targeting and nanoparticle-mediated encapsulation can reduce the toxicity of antitumour drugs and improve their efficacy. Here, we describe the performance of a ...nanotherapeutic encapsulating a hydrolytically sensitive docetaxel prodrug and conjugated to an antibody specific for EphA2-a receptor overexpressed in many tumours. Administration of the nanotherapeutic in mice led to slow and sustained release of the prodrug, reduced exposure of active docetaxel in the circulation (compared with administration of the free drug) and maintenance of optimal exposure of the drug in tumour tissue. We also show that administration of the nanotherapeutic in rats and dogs resulted in minimal haematological toxicity, as well as the absence of neutropenia and improved overall tolerability in multiple rodent models. Targeting of the nanotherapeutic to EphA2 improved tumour penetration and resulted in markedly enhanced antitumour activity (compared with administration of free docetaxel and non-targeted nanotherapeutic controls) in multiple tumour-xenografted mice. This nanomedicine could become a potent and safe therapeutic alternative for cancer patients undergoing chemotherapy.
Due to the myriad interactions between prosurvival and proapoptotic members of the Bcl-2 family of proteins, establishing the mechanisms that regulate the intrinsic apoptotic pathway has proven ...challenging. Mechanistic insights have primarily been gleaned from in vitro studies because genetic approaches in mammals that produce unambiguous data are difficult to design. Here we describe a mutation in mouse and human Bak that specifically disrupts its interaction with the prosurvival protein Bcl-x
L
. Substitution of Glu75 in mBak (hBAK Q77) for leucine does not affect the three-dimensional structure of Bak or killing activity but reduces its affinity for Bcl-x
L
via loss of a single hydrogen bond. Using this mutant, we investigated the requirement for physical restraint of Bak by Bcl-x
L
in apoptotic regulation. In vitro,
Bak
Q75L
cells were significantly more sensitive to various apoptotic stimuli. In vivo, loss of Bcl-x
L
binding to Bak led to significant defects in T-cell and blood platelet survival. Thus, we provide the first definitive in vivo evidence that prosurvival proteins maintain cellular viability by interacting with and inhibiting Bak.
FAS (APO-1/CD95) and its physiological ligand, FASL, regulate apoptotic death of unwanted or dangerous cells in many tissues, functioning as a guardian against autoimmunity and cancer development
1
-
...4
. Distinct cell types differ in the mechanisms by which the ‘death receptor’ FAS triggers their apoptosis
1
-
4
. In type I cells, such as lymphocytes, activation of ‘effector caspases’ by FAS-induced activation of caspase-8 suffices for cell killing whereas in type II cells, including hepatocytes and pancreatic β-cells, amplification of the caspase cascade through caspase-8 mediated activation of the pro-apoptotic BCL-2 family member BID
5
is essential
6
-
8
. Here we show, that loss of X-chromosome linked inhibitor of apoptosis (XIAP)
9
,
10
function by gene-targeting or treatment with a second mitochondria-derived activator of caspases (SMAC
11
, also called DIABLO
12
: direct IAP binding protein with low pI) mimetic drug rendered hepatocytes independent of BID for FAS-induced apoptosis signalling. These results show that XIAP is the critical discriminator between type I versus type II apoptosis signalling and suggest that IAP inhibitors should be used with caution in cancer patients with underlying liver conditions.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Fas ligand (FasL), an apoptosis-inducing member of the TNF cytokine family and its receptor, Fas, are critical for shutdown of chronic immune responses
1
-
3
and prevention of autoimmunity
4
,
5
. ...Accordingly, mutations in their genes cause severe lymphadenopathy and autoimmune disease in mice
6
,
7
and humans
8
,
9
. FasL function is regulated by deposition in the plasma membrane and metalloprotease-mediated shedding
10
,
11
. We generated gene-targeted mice that selectively lack either secreted FasL (ΔsFasL) or membrane-bound FasL (ΔmFasL) to resolve which of these forms is required for cell killing and to explore their hypothetical non-apoptotic activities. Mice lacking sFasL (
FasL
Δs/Δs
) appeared normal and their T cells readily killed target cells, whereas T cells lacking mFasL (
FasL
Δm/Δm
) could not kill cells through Fas activation.
FasL
Δm/Δm
mice developed lymphadenopathy and hyper-gammaglobulinaemia, similar to
FasL
gld/gld
mice, which express a mutant form of FasL that cannot bind Fas, but surprisingly, (on a C57BL/6 background)
FasL
Δm/Δm
mice succumbed to SLE-like autoimmune kidney destruction and histiocytic sarcoma, diseases that occur only rarely and considerably later in
FasL
gld/gld
mice. These results demonstrate that mFasL is essential for cytotoxic activity and constitutes the guardian against lymphadenopathy, autoimmunity and cancer whereas excess sFasL appears to promote autoimmunity and tumorigenesis through non-apoptotic activities.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Several Academic Social Networking Sites (ASNSs) have been launched in the last few years and their number of members is growing. Researchers using ASNSs come from very divergent scientific ...backgrounds and academic levels, prompting one to ask the question, What kind of communication and self‐presentation behaviors occur within these structures? The qualitative study presented in this article analyzed the communication behavior of a selected sample on ResearchGate (RG). It investigates how researchers present themselves on their personal‐profile sites and how they interact with other researchers. Overall, the results show that mostly young male academics without previous connections to each other (for example, faculty colleges) use RG for their scholarly exchanges. In general, communication behavior is characterized by an objective, professional, unemotional choice of words and seldom the use of polite salutations or words of farewell. However, there seems to be a correlation between long discussion and an increased use of colloquial and emotional language. Based on our findings, we derived preliminary practical recommendations for communications on ASNSs to improve the relationships in online academic interactions, to foster inclusiveness of gender and culture, and to reduce insecurity in matters of communication, presentation, and the exchange of scientific data.
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